Gene transfer method

ABSTRACT

A method for efficiently transferring a gene to a target cell is provided. A method of transferring a gene to a target cell, including adding or administering a positively charged complex (A) composed of the gene and a cationic substance and gas-filled microparticles (B) to a target cell-containing composition or a living body and then exposing the target cell-containing composition or the living body to a low-frequency ultrasound.

TECHNICAL FIELD

The present invention relates to a method of and a kit for efficientlytransferring a gene to a target cell in in vivo and in vitro.

BACKGROUND ART

As methods of transferring a gene to a target cell, for example, amethod of administering a gene enclosed in quaternary ammoniumsalt-containing liposomes (Non-Patent Document 1) and a method ofadministering a gene in conjunction with protamine or the like(Non-Patent Document 2) are known. However, these methods are notsatisfactory yet in their transfer efficiency of a gene to a targetcell.

In addition, it is known that a gene can be transferred to a target cellby administering the gene simultaneously with microbubbles made of athin shell of albumin enclosing a propane octafluoride gas or the liketherein and exposing the microbubbles to an ultrasound to causecavitation of the enclosed gas (Non-Patent Document 3).

[Non-Patent Document 1] Felgner, P. L. Cationic liposome-mediatedtransfection with lipofection reagent. Meth. Mol. Biol. 1991, 91-98.

[Non-Patent Document 2] Gao, X. and Huang, L., A novel cationic liposomereagent for efficient transfection of mammalian cells. Biochem. Biophys.Res. Commun. 1991, 179, 280-285.

[Non-Patent Document 3] Tachibana, K., Uchida, T., Ogawa, K., Yamashita,N., Tamura, K., Induction of cell-membrane porosity by ultrasound.Lancet 1999, 353, 1409.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

However, the gene transfer efficiency with the above method usingmicrobubbles is still low. Consequently, a method that can achievehigher transfer efficiency has been desired.

Means for Solving the Problems

Accordingly, the present inventors have completed the present inventionby arriving at the fact that the transfer efficiency of a gene to atarget cell can be dramatically improved by 10 to 10000 times of thoseof conventional methods by previously combining the gene and a cationicsubstance into a complex having a positive surface charge and exposingthis complex in conjunction with microbubbles to an ultrasound, insteadof using the gene and the microbubbles as they are.

That is, the present invention provides a method of transferring a geneto a target cell, including adding or administering a positively chargedcomplex (A) composed of the gene and a cationic substance and gas-filledmicroparticles (B) to a target cell-containing composition or a livingbody, and then exposing the target cell-containing composition or theliving body to a low-frequency ultrasound.

The present invention further provides a kit for transferring a gene toa target cell, wherein the kit including a positively charged complex(A) composed of the gene and a cationic substance, and gas-filledmicroparticles (B).

EFFECT OF THE INVENTION

According to the present invention, an objective gene can be transferredto a target cell with significantly high efficiency in both in vitro andin vivo. Therefore, the present invention can increase the productionratio of transformed cells that can not been obtained by conventionalmethods due to their low transfer efficiency. Furthermore, the presentinvention can dramatically increase the efficacy ratio of gene therapy.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is characterized by using a positively chargedcomplex (A) of a gene and a cationic substance. Here, examples of thegene include DNAs, RNAs, antisense DNAS, siRNAs, decoys, and therapeuticoligonucleotides. Examples of the cationic substance include cationicpeptides such as protamine, poly-L-lysine, poly-L-arginine, andornithine; and cationic polymers such as polyethyleneimine, cationicdendrimers, and chitosan. The complex of the gene and the cationicsubstance can be prepared, for example, by mixing the gene and thecationic substance in purified water. Since aggregation may occurdepending on the solvent, a previous examination should be performed. Inaddition, it is necessary that the entire charge of the prepared complexis positive. The charge is preferably adjusted to +5 to +20 mV as thezeta potential. The zeta potential can be measured with a commonly-usedzeta potential analyzer.

The particle diameter of the complex is preferably about 100 to 300 nmfrom the viewpoint of gene transfer efficiency. This particle diametercan be measured with a laser scattering particle analyzer.

The gene and the cationic substance to be used are preferably mixed at aweight ratio of 1:100 to 100:1 and more preferably at a ratio of 1:10 to10:1.

In addition, as the gas-filled microparticles (B), conventionally usedmicrobubbles can be used, for example, such as albumin microspheresenclosing a gas therein and gas-filled liposomes. Examples of knownmicrobubbles include Alubunex (Molecular Biosystems), Levovist(Schering), Sonavist (Schering), Echovist (Schering), Sonazoid(Nycomed), Optison (Nycomed-Amersham), Definity (DuPont Pharmaceutical),and SonoVue (Bracco).

Examples of the gas-filled liposomes include gas-filled liposomes thatare prepared by filling the void space of a sealed container containinga liposome suspension in a volume amounting to 20 to 80% of the innercapacity thereof with a fluoride gas or a nitrogen gas and then exposingthem to an ultrasound.

Examples of lipids used as the membrane constituent of the liposomeinclude phospholipids, glyceroglycolipids, sphingoglycolipids, cationiclipids in which a primary amino group, a secondary amino group, atertiary amino group, or a quaternary ammonium group is introduced intothe above lipids, lipids in which polyalkylene glycols are introducedinto the above lipids, and lipids to which ligands to various types ofcells, tissues and the like are bound.

The phospholipids includes natural and synthetic phospholipids, such asphosphatidylcholines (e. g., soybean phosphatidylcholine, egg yolkphosphatidylcholine, distearoyl phosphatidylcholine, and dipalmitoylphosphatidylcholine), phosphatidylethanolamines (e. g., distearoylphosphatidylethanolamine), phosphatidylserines, phosphatidic acid,phosphatidylglycerols, phosphatidylinositols, lysophosphatidylcholines,sphingomyelins, egg yolk lecithins, soybean lecithins, and hydrogenadded phospholipids.

Examples of the glyceroglycolipids include sulfoxyribosyl glycerides,diglycosyl diglycerides, digalactosyl diglycerides, galactosyldiglycerides, and glycosyl diglycerides. Examples of thesphingoglycolipids include galactosyl cerebrosides, lactosylcerebrosides, and gangliosides.

Examples of the cationic lipids include lipids in which an amino group,an alkylamino group, a dialkylamino group, or a quaternary ammoniumgroup such as a trialkylammonium group, amonoacyloxyalkyl-dialkylammonium group or adiacyloxyalkyl-monoalkylammonium group, is introduced into the abovephospholipids, glyceroglycolipids or sphingoglycolipids. Examples of thepolyalkylene glycol-modified lipids include lipids in which the abovephospholipids, glyceroglycolipids or sphingoglycolipids are modifiedwith polyethylene glycol, polypropylene glycol or the like, such asdi-C₁₂₋₂₄acyl-glycerol-phosphatidylethanolamine-N-PEG.

In addition, a membrane stabilizer such as cholesterols and anantioxidant such as tocopherol, stearylamine, dicetylphosphate organglioside may be used, according to necessity.

Examples of the ligand to a target cell, a target tissue or a targetlesion include ligands to cancer cells, such as transferrin, folic acid,hyaluronic acid, galactose and mannose. In addition, monoclonalantibodies and polyclonal antibodies can be used as the ligand.

The previously prepared liposomes may contain a gene or the liketherein, as long as they have an aqueous phase in the inside.

The liposomes can be produced by a known process for preparingliposomes, for example, by the liposome preparation method of Bangham,et al., (J. Mol. Biol. 1965, 13, 238), an ethanol injection method (J.Cell. Biol. 1975, 66, 621), a French press method (FEBS Lett. 1979, 99,210), a freeze and thawing method (Arch. Biochem. Biophys. 1981, 212,186), or a reverse phase evaporation method (Proc. Natl. Acad. Sci. USA1978, 75, 4194). For example, a liposome suspension is prepared bydissolving a lipid in an organic solvent, adding an aqueous solutionthereto, and then treating the resulting mixture with an ultrasound.Then, if necessary, the suspension is applied to an extruder and/or amembrane filter for particle sizing. In such a case, the particles arepreferably sized to have a particle diameter of 1 μm or less, morepreferably 100 to 800 nm, and particularly preferably 100 to 600 nm.

The prepared liposome suspension is poured in a sealed container. Inthis stage, the void space of the container is preferably 20 to 80%,more preferably 30 to 80%, and particularly preferably 50 to 80% of theinner capacity of the container. When the void space is less than 20%,the induction ratio of a gas into the produced liposomes is too low. Thevoid space exceeding 80% is uneconomical.

This void space is filled with a fluoride gas or a nitrogen gas.Examples of the fluoride gas include sulfur hexafluoride andperfluorohydrocarbon gases, such as CF₄, C₂F₆, C₃F₈, C₄F₁₀, C₅F₁₂, andC₆F₁₄. Among them, C₃F₈, C₄F₁₀, and C₅F₁₂ are particularly preferred. Inaddition, a nitrogen gas can also be used. The pressure of the filledgas is preferably 1 atmosphere (gauge pressure) or more and particularlypreferably 1 to 1.5 atmospheres. A simple way for filling the void spacewith a gas is injection, for example, with a needle syringe through arubber stopper. An injection cylinder may also be used.

Subsequently, an ultrasound treatment is conducted. For example, thecontainer may be exposed to an ultrasound of 20 to 50 kHz for 1 to 5minutes. With this ultrasound treatment, the aqueous solution in theliposomes is replaced with a fluoride gas or a nitrogen gas to givegas-filled liposomes. The given gas-filled liposomes have a particlediameter approximately the same as that of the raw liposomes.Accordingly, the gas-filled liposomes having a particle diameter withina certain range, e.g., 1 μm or less, more preferably 50 to 800 nm, andparticularly preferably 100 to 600 nm, can be readily produced by sizingthe raw liposomes when they are prepared.

Furthermore, the gas-filled liposomes can be readily produced at a site,such as a hospital, only by conducting an ultrasound treatment, if asealed container containing a liposome suspension and filled with afluoride gas or a nitrogen gas is previously prepared and supplied tothe hospital or the like.

The gas-filled liposomes thus obtained can have a small particlediameter and a constant particle size distribution, and can be deliveredto a microvasculature, a deep tissue or the like.

Furthermore, in the present invention, the above complex (A) may beenclosed in the gas-filled microparticles (B). The process for enclosingthe complex into the microparticles may be conducted during the step ofpreparing the gas-filled microparticles, or may be performed after thepreparation of the gas-filled microparticles by adding the complex (A)to the microparticles and mixing them.

In the present invention, the above complex (A) and the gas-filledmicroparticles (B) are added or administered to a target cell-containingcomposition or a living body. Examples of the target cell-containingcomposition include target cell culture solutions. Examples of theliving body include mammals including human, birds, fishes, reptiles,insects, and plants. The target cell includes a cell into which a geneis introduced or a tissue including such a cell.

In a case of in vitro, the above complex (A) and the gas-filledmicroparticles (B) are added to a target cell culture solution and themixture is exposed to a low-frequency ultrasound. In a case of in viva,the above complex and the gas-filled microparticles are administered toa living body, followed by exposing the living body to diagnosticultrasound (2 to 6 MHz) to confirm the delivery of the complex and themicroparticles to the target cells. Once the delivery is confirmed, alow-frequency ultrasonication is conducted. The administration may betopical administration or intravenous administration.

Exposing the gas-filled microparticles (B) to a low-frequency ultrasoundcontaining a resonance frequency of 0.5 to 2 MHz leads to disruption ofthe microparticles and cavitation caused by microbubbles of the gas. Asa result, the above complex (A) or the above complex (A) in thegas-filled microparticles present near the cavitation site isefficiently introduced into the target cells. The mechanism that thecomplex (A) is efficiently induced into the target cells is unclear, butis assumed that the complex can be readily brought into contact with thecell surfaces due to its positive charge.

EXAMPLE

The present invention will hereinafter be described in detail withreference to the example, but is not limited thereto.

Abbreviations used in the example are as follows:

DPPC: dipalmitoyl phosphatidylcholine

DOPE: dioleoyl phosphatidylethanolamine

DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane

Example 1

(1) Plasmid DNAs coding luciferase were mixed with protamine to preparea DNA-protamine complex followed by reducing the size thereof. Theentire charge of the complex was adjusted to be positive (+0.5 to +20 mVof zeta potential) For comparison, the complex having negative entirecharge (−7 mV of zeta potential) was also made.

(2) DPPC Liposome

Lipids of DPPC and cholesterol (1:11 (m/m)) were dissolved in an organicsolvent mixture of chloroform and isopropyl ether (1:1, v/v), and anaqueous solution such as saline (or an aqueous solution containing adrug) was added thereto in a volume amounting to a half of the organicsolvent (i.e., chloroform : isopropyl ether aqueous solution=1:1:1,v/v). The resulting mixture was mixed to give an emulsion. The emulsionwas subjected to a reverse phase evaporation method (REV method) toprepare liposomes. The liposomes were sized by passing them throughpolycarbonate membranes of 400 nm, 200 nm and 100 nm with an extruder.

(3) DOTAP Liposome

DOTAP and DOPE (1:1, (w/w)) were dissolved in chloroform, and themixture was put in a pear-shaped flask. The organic solvent wasevaporated while rotating with a rotary evaporator to produce a thinfilm of a lipid on the wall (production of a lipid film). Then,hydration was conducted using a solvent such as saline to produceliposomes. The liposomes were reduced in size by an ultrasound treatmentor by passing them through polycarbonate membranes of 400 nm, 200 nm and100 nm with an extruder.

(4) The following reagents were used as commercially availablegene-delivering reagents composed of cationic liposomes:

Lipofectin™ (DOTMA:DOPE=1:1, w/w), and

LipofectACE™ (DDAB:DOPE=1:1.25, w/w).

(5) Enclosure of Perfluoropropane Gas

A liposome aqueous solution (lipid concentration: 5 mg/mL) was put in avial (5 mL, 10 mL, or 20 mL, for example) in a volume amounting to 30%of the capacity of the vial (1.5 mL, 3 mL, or 6 mL). Perf luoropropanegas was put into the vial to replace for air therein. The vial wassealed with a rubber stopper, and perfluoropropane was further addedthrough the rubber stopper with a needle syringe to the volume of 1.5times of the inner capacity, so that the inner pressure became about 1.5atms. A bath-type ultrasound apparatus (42 kHz) was filled with water,and the vial was left standing therein and exposed to an ultrasound forone minute.

(6) AsPC-1 cells (4×10⁴ cells/well) were cultured in a 48-well plate.The DNA-protamine complex (1 μg of DNA, lipid:DNA=12:1, w/w) and thegas-filled PEG-liposomes were added thereto and then exposed to a pulsedultrasound of 1 MHz for three seconds. The culture solution wasimmediately washed three or four times repeatedly. After addition of aculture medium, the cells were further cultured for two days. Then,luciferase activity was measured by a conventional method. The resultsare shown in Table 1.

TABLE 1 Charge state Luciferase Perfluoropropane of activity gas-filledDNA/protamine Ultrasound (RLU/mg liposome complex treatment protein)DPPC LP positive 0.6 × 10³ charge DPPC LP positive +SONIC 6.3 × 10³charge DPPC LP negative 0.1 × 10³ charge DOTAP LP positive 4.9 × 10⁶charge DOTAP LP positive +SONIC 205 × 10⁶ charge DOTAP LP negative 0.1 ×10⁶ charge Lipofectin ™ positive 0.1 × 10⁶ charge Lipofectin ™ positive+SONIC 129 × 10⁶ charge LipofectACE ™ positive 0.3 × 10⁶ chargeLipofectACE ™ positive +SONIC 135 × 10⁶ charge

It was indicated from the results that the high expression level wasachieved when perfluoropropane gas-filled cationic liposomes and thepositively charged DNA/protamine complex were exposed to a low-frequencyultrasound.

1. A method of transferring a gene to a target cell, comprising addingor administering a positively charged complex (A) composed of the geneand a cationic substance and gas-filled microparticles (B) to a targetcell-containing composition or a living body, and then exposing thetarget cell-containing composition or the living body to a low-frequencyultrasound.
 2. The method of transferring a gene according to claim 1,wherein the target cell-containing composition is a target cell culturesolution.
 3. The method of transferring a gene according to claim 1 or2, wherein the cationic substance is a cationic peptide or a cationicpolymer.
 4. The method of transferring a gene according to any one ofclaims 1 to 3, wherein the gas-filled microparticles are microspheres ofa polymer or liposome enclosing a gas therein.
 5. The method oftransferring a gene according to any one of claims 1 to 4, wherein thepositively charged complex (A) composed of the gene and a cationicsubstance is enclosed in the gas-filled microparticles (B).
 6. A kit fortransferring a gene to a target cell, comprising a positively chargedcomplex (A) composed of the gene and a cationic substance, andgas-filled microparticles (B).
 7. The kit according to claim 6, whereinthe kit is used for adding the positively charged complex (A) composedof a gene and a cationic substance, and gas-filled microparticles (B) toa target cell-containing composition and then exposing the targetcell-containing composition to a low-frequency ultrasound.
 8. The kitaccording to claim 6, wherein the kit is used for administering thepositively charged complex (A) composed of a gene and a cationicsubstance and gas-filled microparticles (B) to a living body and thenexposing the living body to a low-frequency ultrasound.
 9. The kitaccording to claim 6 or 8, wherein the target cell-containingcomposition is a target cell culture solution.
 10. The kit according toany one of claims 6 to 9, wherein the cationic substance is a cationicpeptide or a cationic polymer.
 11. The kit according to any one ofclaims 6 to 9, wherein the gas-containing microparticles aremicrospheres of a polymer or liposome enclosing a gas therein.
 12. Thekit according to any one of claims 6 to 11, wherein the positivelycharged complex (A) composed of a gene and a cationic substance isenclosed in the gas-filled microparticles (B).